Proline accumulation is a common response of plants to different biotic and abiotic stresses. In the model legume Lotus japonicus, osmotic stress-induced proline accumulation is one of the first responses of the plant, converting proline in a reliable stress marker. The main biosynthetic pathway of proline is from glutamate and the reaction catalyzed by the enzyme Δ1-pyrroline 5-carboxylate synthase (P5CS) is the rate limiting step. L. japonicus has been suggested to have three different P5CS genes. Here the predicted P5CS genes of L. japonicus were analyzed in silico and their expression under osmotic stress was determined. Contrary to previous suggestions this study demonstrated that L. japonicus has two different P5CS genes, as most dicotyledonous plants do. The gene that is inducible by osmotic stress and is located on chromosome 1, was called LjP5CS1, and the one located on chromosome 2 and not inducible by osmotic stress was called LjP5CS2.